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Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the K half of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival., Competing Interests: Conflict of interest G. T. M. is the founder of Nexomics Biosciences Inc. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF K for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the half for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.K half for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.